Melamine derivatives for use in the treatment of cancer

ABSTRACT

A compound of the formula: ##STR1## wherein each R 1 , which may be the same or different, is hydrogen, alkyl, or an electron withdrawing group; and 
     R 2  is hydrogen, alkyl, or an electron withdrawing group.

This application is a 371 of PCT/GB93/00625, filed Mar. 26, 1993.

This invention relates to novel 2,4,6-triamino- 1,3,5-triazines,compositions containing them, processes for making them and their use inthe treatment of carcinomas, particularly ovarian carcinomas.

Trimelamol [2,4,6-tris{(hydroxymethyl) (methyl) amino)-1,3,5-triazine]is clinically active, particularly against ovarian carcinomas, but itsclinical development has been halted due to difficulties withformulation due to instability with respect to the formation of dimersduring formulation. It has been established that the half-life oftrimelamol activity in humans is short and that may limit its clinicalefficacy (I. R. Judson, et al Cancer Res. 49, 5475-5479, 1989). Webelieve that this is, in part, due to the chemical instability of theN-hydroxymethyl functions resulting in the release of formaldehyde. Wehave investigated reducing the number of N-hydroxymethyl functions andstabilizing these functions using electron-withdrawing organic groups(defined in the present context as electron-withdrawing relative tomethyl), with a view to lengthening the half-life and also improvingamenability to formulation, for example in aqueous solutions.

Accordingly this invention provides novel 2,4,6-triamino-1,3,5-triazineshaving the following general formula: ##STR2## wherein each R¹ which maybe the same or different, is hydrogen, alkyl or an electron-withdrawinggroup and R² is hydrogen, alkyl or an electron-withdrawing organicgroup. Preferably, all three groups R¹ are not hydrogen. The alkyl groupR¹ and/or R² is preferably a C₁ -C₄ alkyl group, particularly methyl andit is preferred that all three R¹ groups, when alkyl, are all methyl.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred electron-withdrawing organic groups are --CH₂ CF₃ and --CH₂.tbd.CH. Because of the greater stability conferred on such compounds bythe presence of such electron withdrawing substituents, which mayconstitute in lengthening the half-life and also in improvingamenability to formulation, they may be prepared by allowingtris-hydroxymethyl compounds or precursors thereof to decompose inaqueous organic media and separating from the mixture of products (seeFIG. 1) thus generated the appropriate compounds of the presentinvention, for example by chromatography on silica gel.

We have found that these new analogues of trimelamol have a similarlevel of activity against carcinomas, particularly ovarian carcinomas,as trimelamol, but are more stable and do not form dimers and polymersand thus are more amenable to formulation.

The compounds of the present invention are also prepared via novelintermediate compounds of the general formula: ##STR3## wherein R¹ andR² are as defined above for the formula I

The intermediates are prepared by reacting a cyanuric halide of generalformula: ##STR4## wherein X is fluoro or chloro with an amine of theformula R¹ --NH₂ or R¹ R² NH₂, wherein R¹ and R² are as defined informula (I), optionally in the presence of caesium fluoride.

In the absence of caesium fluoride, less than three of the substituentson the 1,3,5-triazine ring may be displaced, which allows for thepreparation of asymmetrical compounds.

Treatment of the intermediates II with aqueous formaldehyde, optionallyin the presence of potassium carbonate, gives the compounds of formula(I). In order to provide compounds of the formula I in which R¹ ismethyl and R² is hydrogen, starting from compounds of the formula II inwhich R¹ and R² are also methyl and hydrogen respectively, we prefer touse a concentration of formaldehyde of from about 2 to 5% (w/v), forexample about 3% (w/v). This provides a final product which contains asthe major product the compound of the formula I. A small amount of thecorresponding trimelamol (i.e. R¹ =methyl, R² =CH₂ OH) and `monomelamol`(i.e. three methyls but only one hydroxymethyl group) compounds will beproduced. The presence of these compounds does not significantly affectthe activity of the preparation of the compound of the invention inbiological assays. However, if desired, the purity of the preparationmay be increased by recrystallisation. For example, the material may bedissolved in methanol-water (e.g. at a ratio of 9:1), andrecrystallised.

The compounds of this invention are biologically active and are of useagainst ovarian carcinomas, particularly against cisplatin-resistantovarian carcinomas.

Also included within the scope of the present invention arepharmaceutical compositions which comprise, as active ingredient, atleast one compound of general formula I, in association with apharmaceutically acceptable carrier or diluent.

The compounds of the invention will normally be administered orally orby injection.

Compositions for parenteral administration will normally be solutions inaqueous saline, which is pyrogert free for human use. Such compositionscan be administered intravenously or intraperitoneally.

Compositions for oral administration will mostly be in solid or liquidform, mostly as tablets, capsules, lozenges, etc. Liquid compositionscan be solutions or dispersions in aqueous or non-aqueous media. Idealsolutions are of neutral or alkaline pH and of low ionic strength e.g.5% dextrose.

Suitable daily doses of the compounds of the invention in therapeutictreatment of the human or animal body range from about 100 mg to 3 g/m²body-surface.

The following Examples illustrate the preparation of the compounds ofthe present invention.

EXAMPLE 1

2,4-Bis[(hydroxymethyl) (methyl) amino]-6-methylamino-1,3,5-triazine

To a 3% w/v aqueous solution of formaldehyde (15 ml) was added potassiumcarbonate (691 mg, 5 mmol) then trimethylmelamine (841 rag, 5 mmol). Thereaction mixture was stirred at room temperature until the initiallyclear solution (pH 11.5) became cloudy (2-3 h) then set aside overnight(16 h). The white granular solid which separated was recovered byfiltration, washed with water (4×5 ml) and the product dried in vacuoover anhydrous CaCl₂. Yield 593 mg (52%); ¹ H-NMR spectrum δ_(H) (Me₂SO--d₆) 2.75 (app d, 3, HNCH₃), 4.99 (br s, 4, HOCH₂), 5.36 (br s, 2,OH) 6.61 (br s, 1, NH); mass spectrum (FAB; glycerol/thioglycerolmatrix) m/z 229 ([M+H]⁺, 70%), 211 (229--H₂ O, 100%), 199 (229--CH₂ O,35%), 181 (199--H₂ O, 50%), 169 (199--CH₂ O, 30%). Anal. C₈ H₁₆ N₆ O₂requires C, 42.10; H, 7.07; N, 36.82: found C, 41.87; H, 7.01; N,36.55%.

In the Examples which follow, this compound is referred to as CB7646.

EXAMPLE 2

Further purification of title compound of Example 1.

Using the procedures described in Example 1 above, but with 10 times theamount of starting materials, 6.325 g of product was obtained. HPLCanalysis revealed the preparation to have the following composition:

title compound: 65%, trimelamol 22%, monohydroxymethyl derivative 12%.

This material (3 g) was dissolved in methanol-water, 9:1 (100 ml) at 37°C. and cooled at -20° C. for 24 h. The white crystalline solid wasrecovered by rapid filtration and dried in vacuo over anhydrous CaCl₂ togive 1.37 g of material having the following composition: title compound87% trimelamol 4%, monohydroxymethyl derivative 9%. Signals in the ¹H-NMR spectrum (D₂ O, determined at 37° C.) were: title compound δ3.08(HNCH₃), 3.30 (HOCH₂ NCH₃), 5.29 HOCH₂); trimelamol 3.33 and 5.32;monohydroxymethyl derivative 3.05, 3.27 and 5.26.

EXAMPLE 3

2,4-Bis[(hydroxymethyl)(2,2,2-trifluorethyl)amino]-6-(2,2,2-trifluoroethyl)amino-1,3,5-triazine

A solution of2-[([hydroxymethoxy]methyl)(2,2,2-trifluoroethyl)amino]-4,6-bis(hydroxymethyl) (2,2,2-trifluoroethyl) amino-1,3,5-triazine (500 mg,1.02 mmol) in a mixture of acetone (3 ml) and water (2 ml) was set asideat room temperature for 18 h. Acetone was removed under vacuum and theorganic materials were extracted with diethyl ether.

The organic phase was concentrated and applied to a column (50 g, 3 cmdia.) of silica gel (Merck, Art. No. 9385) which was eluted with diethylether. There was successively eluted2-[(hydroxymethyl)(2,2,2-trifluoroethyl)amino]-4,6-bis[2,2,2-trifluoroethyl)amino]-1,3,5-triazine (23 mg), the title compound(144 rag, 33% yield) and 2,4,6-tris [(hydroxymethyl)(2,2,2-trifluoroethyl) amino]1,3,5-triazine (111 mg). The title compoundis obtained as a white solid by trituration of the appropriate fractionswith ice-cold water, recovery by filtration and desiccation in vacuoover calcium chloride. NMR spectrum: δ_(H) (Me₂ SO--d₆) 4.09 (brq, 2, F₃CCH₂ NH), 4.41 (brq. 4, F₃ CCH₂ NCH₂ OH) 5.06 (d, 4J=7.1 Hz, CH₂ OH),5.78 (brs, 2, OH), 7.80 (brs, 1, NH) δ_(F) -70.23, -70.03 (2s, 3, F₃CCH.sub. 2 NH) -68.3 (s, 6, F₃ CCH₂ NCH₂ OH).

In the Examples which follow, this compound is referred to as CB7683.

EXAMPLE 4

Stability of Compounds of the Invention

(i) Stability in Solution

Compounds were dissolved in DMSO to a concentration of 50 mM. Aliquotswere then dispersed into the appropriate medium to give a finalconcentration of 100 μM in a volume of about 10 ml. The dilutedpreparations of trimelamol and CB7646 (see Example 1) for HPLC analysiswere stored in a water bath at 21°-24° C. (to simulate room temperature)or at 37° C. in water, 0.9% NaCl or 5% dextrose. Aliquots were removedfrom each preparation at intervals to assess their stability (i.e.half-life, T^(1/2)) which was measured using HPLC analysis. Thisentailed an isocratic elution using a mobile phase comprising 10%acetonitrile, 90% 0.05M ammonium bicarbonate. The 15 cm column waspacked with C8 octyl Spherisorb material. The column was encased in acooling cabinet which was maintained at 14°-17° C. Standards of freshlyprepared solutions were run throughout the analysis period by way ofcontrols.

T^(1/2) measurements were made by measurement of the disappearance ofcompound by decreasing peak area with time, using a Data System 450MT2data acquisition system (Kontron Instruments, Watford, UK) linkeddirectly to the detector on the HPLC system (set at 225 nM). T^(1/2)measurements were read from a semi-logarithmic plot of peak area (y)versus time (x).

The results are shown in table 1 and indicate that CB7646 has superiorstability.

                  TABLE 1                                                         ______________________________________                                        Compound   Medium         °C.                                                                          T.sup.1/2  (Min)                              ______________________________________                                        Trimelamol deionised water                                                               pH 7.5         37    120                                                      0.9% NaCl,                                                                    pH 4.9         r.t.  273                                                      5% Dextrose,                                                                  pH 4.0         r.t.  348                                           CB7646     Deionised water                                                               pH 7.5         37    180                                                      Deionised water                                                               pH 7.5         r.t.  1080                                                     0.9% NaCl,                                                                    pH 5.0         r.t.  960                                                      5% Dextrose                                                                   pH 4.0         r.t.  1320                                          ______________________________________                                    

(ii) Dimer/Polymer Formation in Solution

An aqueous solution of CB7646 and trimelamol in 4 ml aliquots at aconcentration of 4-5 mg/ml was left to stand overnight (14-16 hours) atroom temperature. By the end of this period, the trimelamol solution hadformed a heavy precipitate, indicative of dimer and polymer formation.Similar polymerisation of trimelamol over a period of time provedproblematic during its Phase I and II clinical trials (Judson et al,1989, Cancer Res. 49;5475-5479; Judson et al, 1991, Br. J. Cancer 63;311-313). In contrast, preparations of CB7646 prepared in Examples 1 and2 did not form a precipitate, indicating the monomeric form is morestable that trimelamol.

EXAMPLE 5

Cytotoxicity of Compounds of the Invention

The cytotoxicity of CB7646 and CB7683 was compared with trimelamolagainst mammalian tumour cell lines using the MTT assay. This assay isbased upon the selective ability of living but not dead cells to reducethe tetrazolium salt MTT (3-[4,5-dimethylthiazol-2-yl]-25-diphenyltetrazolium bromide) to purple formazan (Mosmann et al, 1983, J. Immun.Methods 65; 55-63; Carmichael et al (1987) Cancer Res. 47; 936-942).Cell lines were grown in culture with continual drug exposure. The IC₅₀values (in μm) of the compounds (i.e. concentration giving 50%inhibition of cell growth as compared with untreated control) weredetermined, and are shown in Table 2. Figures in parenthesis refer tostandard deviation or +/- values for 2 or more measurements.

                  TABLE 2                                                         ______________________________________                                        CELL LINE  TRIMELAMOL    CB 7646   CB 7683                                    ______________________________________                                        PC6        12.9 (2.7)    25.1 (2.9)                                                                              31.6 (1.0)                                 WALKER 256 9.4 (0.5)     10.7 (0.2)                                                                              ND                                         H69        8.5 (2.3)     14.7 (4.9)                                                                              8.9 (1.1)                                  CH1        23.4 (4.4)    35.8 (13.1)                                                                             40.9 (12.0)                                ______________________________________                                         (ND  not done).                                                               Cell lines used:                                                              PC6  murine plasmacytoma                                                      Walker 256  rat mammary carcinoma                                             H69  human small cell lung cancer                                             CH1, 41M  human epithelial ovarian cancer                                

The tests on Walker 256 and H69 cells were repeated using a preparationof CB7646 prepared by the recrystallisation method of Example 2. Theresults were:

Walker 256 - 10.5

H69 - 16.5

EXAMPLE 6

Antitumour Activity Towards the ADJ/PC6 Tumour in Mice

The anti-tumour activity of CB 7646 prepared in accordance with Example1 against ADJ/PC6 tumour in mice were compared with that of trimelamol.An implant of 1 mm³ of tumour was made on day 1. On day 20. animalsbearing tumours of comparative size were placed into groups of 4 andtreated with drug on 5 consecutive days, and then left until day 30.Tumours from the treated and controls were dissected and weighed as ameasure of tumour growth. Compounds were given in 5% DMSO/dextrose.

                  TABLE 3                                                         ______________________________________                                        % Inhibition at various Doses                                                 (Tumour wt as % of Control Value)                                             Compound                                                                              Dose (mg/kg)                                                          ______________________________________                                                3.25    6.25     12.5  25    50    100                                CB7646  5.6     -1.0     13.7  5.6   76.8  98.0                               Trimelamol                                                                            0       18.2     13.7  45.5  83.9  96.0                               ______________________________________                                         For CB7646 (dimelamol) the results give LD.sub.50 >100 mg/kg, ED.sub.90 7     mg/kg Therapeutic Index (TI) >1.4                                        

EXAMPLE 7

Example 6 was repeated to obtain more precise LD₅₀ values. The LD₅₀,ED₉₀ and T. I. values were calculated and shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        COMPOUND   LD.sub.50 MG/KG                                                                            ED.sub.90 MG/KG                                                                            T.I.                                     ______________________________________                                        TRIMELAMOL 70           24           2.9                                      CB 7646    142          31           4.6                                      ______________________________________                                    

EXAMPLE 8

CB7646 was tested in vivo against ovarian cancer xenografts of the PXN65cell line transplanted into mice, substantially in accordance withHarrap et al, Annals of Oncology, 1990, 1;65-76. PXN65 is acisplatin-sensitive line. Mice treated with either trimelamol or CB7646showed tumour regression within 28 days whereas in untreated controlstumour growth was uncontrolled, leading to death. The results aresummarised in Table 5.

                  TABLE 5                                                         ______________________________________                                        Activity in vivo against PXN65 Xenografts                                                Dose                                                               COMPOUND   mg/kg    No. Doses GD Days Deaths                                  ______________________________________                                        TRIMELAMOL 30       5         >273    0                                                  15       20        >170    0                                       CB7646     15       20        >140    0                                       ______________________________________                                         GD = Growth delay.                                                       

The data show that CB7646 has a comparable efficacy to trimelamol.

We claim: 1.2,4-Bis[(hydroxymethyl)(methyl)amino]-6]methylamino-1,3,5-triazine.
 2. Amethod for the treatment of ovarian cancer which comprises administeringto a patient in need of treatment an effective amount of a compoundaccording to claim
 1. 3. A pharmaceutical composition comprising thecompound of claim 1 and an inert diluent or carrier.
 4. A method for thetreatment of ovarian cancer which comprises administering to a patientin need of treatment an effective amount of a composition according toclaim
 3. 5. The method of claim 2 or 4 wherein the ovarian cancer iscisplatin resistant ovarian cancer.
 6. A process for the preparation of2,4-Bis[(hydroxymethyl)(methyl)amino]-6]methylamino-1,3,5-triazine whichcomprises reacting trimethylmelamine with formaldehyde, the formaldehydebeing at a concentration of from about 2% to 5% (w/v), and recoveringsaid 2,4-Bis[(hydroxymethyl)(methyl)amino]-6]methylamino-1,3,5-triazine.7. The process of claim 6, wherein the reaction is performed in thepresence of potassium carbonate.
 8. The process of claim 6, wherein theformaldehyde is used at a concentration of about 3% (w/v).
 9. Theprocess of claim 6, which further comprises a recrystallisation step.